Multilocus variable-number tandem-repeat analysis for genotyping of Shigella sonnei strains isolated from pediatric patients

Aim: The aims of this study were to characterize Iranian Shigella sonnei strains isolated from pediatric cases and evaluate the utility of multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) for genotyping of local S. sonnei strains. Background: S. sonnei has become the dominant species in certain parts of Iran. Although PFGE is still a gold standard for genotyping and source tracking of food-borne pathogens, it is laborious, expensive, time-consuming, and often difficult to interpret. However, MLVA is a PCR-based method, which is rapid, relatively inexpensive and easy to perform. Patients and methods: A total of 47 S. sonnei isolates were obtained from sporadic cases of pediatric shigellosis in Tehran, Iran, during the years 2002-2003 (n=10) and 2008-2010 (n=37). The patients suffered from acute diarrhea and had evidence of more than three episodes of watery, loose, or bloody stools per day. A MLVA scheme based on 7 VNTR loci was established to assess the diversity of 47 S. sonnei isolates. Results: Based on the results, it was clear that the S. sonnei isolates were heterogeneous. Overall, 47 S. sonnei isolates were discriminated into 21 different genotypes. Analysis of the MLVA profiles using a minimum spanning tree (MST) algorithm showed the usefulness of the MLVA assay in discriminating S. sonnei isolates collected over different time periods. However, no correlation was found between the MLVA genotypes and age, gender or clinical symptoms of the patients. Conclusion: It is assumed that our S. sonnei isolates are derived from a limited number of clones that undergo minor genetic changes in the course of time. The present study has provided some valuable insights into the genetic relatedness of S. sonnei in Tehran, Iran.


Introduction
1 Shigellosis, also known as bacillary dysentery or Marlow's Syndrome, continues to be a major cause of mortality and morbidity, especially in children with diarrhea in developing countries where there is overcrowding and poor sanitation (1). The genus Shigella comprises four species: S. dysenteriae, S. flexneri, S. boydii, and S. sonnei. With the exception of S. sonnei, each species may be further divided into several serotypes. Historically, S. sonnei is the predominant Shigella spp. in developed countries. However, recently, a change in trend has been reported from developing countries, where S. flexneri serotypes have been replaced by S. sonnei in areas undergoing ORIGINAL ARTICLE economic development and improvements in hygiene (2)(3)(4). Microbial genotyping is frequently applied for epidemiological investigations and provides useful information for establishing the genetic relatedness among pathogenic strains (5). In this regard, a number of genotyping methods have been developed for S. sonnei, including pulsedfield gel electrophoresis (PFGE) (6,7,8), multilocus enzyme electrophoresis (MLST) (9), multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) (10,11,12,13,14), ribotyping (2,15,16,17), plasmid profiling (5,18), Repetitive Sequence-Based PCR (REP-PCR), and Enterobacterial Repetitive Intergenic Consensus sequence-based PCR (ERIC-PCR) (9,19,20). Among these methods, PFGE is still a gold standard for molecular subtyping and source tracking of food-borne pathogens. However, it is too discriminatory for clonal analysis of S. sonnei strains which have evolved over a longer time span (21). In addition, PFGE is laborious, expensive, time-consuming, often difficult to interpret, and requires rigorous standardization. Moreover, it needs experienced personnel in order to achieve reliable, consistent, and reproducible results. In contrast to PFGE, MLVA is a PCR-based genotyping method, which is rapid, relatively inexpensive and easy to perform. The method is based on the inherent variability of short sequences, which are organized as tandem repeats at multiple VNTR loci (12, 21). Although S. sonnei is becoming an important etiologic agent of pediatric shigellosis in Iran (2,3), there is limited information on the genetic background of the local strains. Therefore, we aimed to characterize S. sonnei isolates using a simple MLVA assay to evaluate the utility of this method for establishing phylogenetic relationships among S. sonnei strains in Iran, Tehran.

Bacterial strains
A total of 47 S. sonnei isolates were obtained from 950 patients (less than 12 years-of-age). These strains were isolated from sporadic cases of endemic shigellosis in Tehran, Iran, during the years 2002-2003 (n=10) and 2008-2010 (n=37). The children suffered from acute diarrhea and had evidence of more than three episodes of watery, loose, or bloody stools per day. Each strain was excluded from one patient. Shigellosis is confirmed through the culture of a stool specimen or rectal swab according to standard laboratory procedures. Briefly, the culture plates (MacConkey agar or XLD agar) were incubated overnight at 37°. Nonlactose fermenting colonies were selected and subjected to routine biochemical and serological tests. The serological test was carried out with commercial antisera (Mast Diagnostic, Merseyside, UK) by using slide agglutination method (2). The verified isolates were preserved at -70°C in Tripticase soy broth with 25% (v/v) glycerol for further analysis. The S. sonnei isolates were not repeatedly subcultured before this study to avoid any possible changes in the number of repeats within VNTR loci.
All ethical issues were considered. Life, health, dignity, integrity, right to self-determination, privacy, and confidentiality of personal information of research subjects were protected in this study.

DNA preparation
A pure culture of S. sonnei was plated on nutrient agar and incubated overnight at 37°C. A single colony was removed from the plate, suspended in 200 μl of sterile deionized water, and boiled for 15 min. After centrifugation at 8,000 g for 6 min, the supernatant was transferred into a new tube for subsequent PCR analysis.

Data analysis
The number of repeats can be easily deduced from the amplicon sizes by manual reading (Figure 1). Amplicon sizes were converted into numbers of repeats based on the formula: number of repeats (bp) = PCR product size (bp) − flanking regions (bp)/repeat size (bp). Repeat numbers were imported into Microsoft Excel. The minimum spanning tree (MST) was constructed with a categorical coefficient based on allelic profiles of the S. sonnei strains using trial version of Ridom MLVA compare software (Ridom® GmbH, Germany). MST is a convenient complementary tool to cluster multiple isolates and visualize the relative diversity within different lineages. A dendrogram of genetic relationships was also generated using the unweighted pair group method with arithmetic averages (UPGMA) method (23).
MLVA based on seven VNTR loci was performed to characterize the S. sonnei strains. Overall, the 47 S. sonnei strains were     They also observed that S. sonnei isolates with no epidemiological linkage were clustered together which may be due to travel within the country and/or person-to-person spread of a particular strain over a long period of time with minor genetic changes (10). Moreover, they reported that there was no clear demarcation of the strains isolated from different years, which was almost in contrast with our results. In the current study, we showed the usefulness of our MLVA assay in discriminating S. sonnei strains collected over different time periods. In Japan, MLVA was also successfully applied to study the epidemiology of S. sonnei strains isolated from cases associated with foreign travel, and the correlations among molecular types, biotypes, resistance types and their geographical areas of origin. Interestingly, it was shown that S. sonnei isolates were classified into different clusters mainly on the basis of their countries of origin (13 sonnei is less discriminatory than those of other studies (10,11,12,13,21), other schemes require a high precision of DNA length measurement, such as microcapillary electrophoresis and fluorescent markers. It is also worth mentioning that developing countries have limited accessibility to such equipments and their expenditures would be a major obstacle for many laboratories (22,25). In our MLVA assay, we selected VNTR markers which can be easily analyzed by eye on agarose gels. Therefore, the assay can be carried out in a laboratory equipped with simple molecular biology equipment. On the other hand, VNTR loci have a wide range of evolutionary rates; thus, they can be exploited to investigate genetic relationships among isolates that have evolved over different timescales. Rapidly evolving VNTR loci with high variability are suitable for discerning closely related isolates or investigation of short-term epidemiology, such as disease outbreaks and disease surveillance, whereas slowly evolving VNTR loci with low variability are appropriate for establishing clearer clonal relationships among strains that have evolved over longer timescales (26).
In conclusion, it is assumed that our S. sonnei strains are derived from a limited number of clones that undergo minor genetic changes in course of time. However, a larger sample size from a variety of geographical regions will be needed to determine which loci provide sufficient resolution for disease surveillance and outbreak investigation. Furthermore, due to monomorphic nature of S. sonnei (27), a larger set of VNTR loci would be more favorable to obtain a clearer separation of clonal groups. Finally, this study has provided some valuable insights into the genetic relatedness of S. sonnei in Tehran, Iran.